Jonathan J. Lee

Melanoma Epigenetics: Novel Mechanisms, Markers, and Medicines

The incidence and mortality rates of cutaneous melanoma continue to increase worldwide, despite the deployment of targeted therapies. Recently, there has been rapid growth and development in our understanding of epigenetic mechanisms and their role in cancer pathobiology. Epigenetics—defined as the processes resulting in heritable changes in gene expression beyond those caused by alterations in the DNA sequence—likely contain the information that encodes for such phenotypic variation between individuals with identical genotypes. By altering the structure of chromatin through covalent modification of DNA bases or histone proteins, or by regulating mRNA translation through non-coding RNAs, the epigenome ultimately determines which genes are expressed and which are kept silent. While our understanding of epigenetic mechanisms is growing at a rapid pace, the field of melanoma epigenomics still remains in its infancy. In this Pathology in Focus, we will briefly review the basics of epigenetics to contextualize and critically examine the existing literature using melanoma as a cancer paradigm. Our understanding of how dysregulated DNA methylation and DNA demethylation/hydroxymethylation, histone modification, and non-coding RNAs affect cancer pathogenesis and melanoma virulence, in particular, provides us with an ever-expanding repertoire of potential diagnostic biomarkers, therapeutic targets, and novel pathogenic mechanisms. The evidence reviewed herein indicates the critical role of epigenetic mechanisms in melanoma pathobiology and provides evidence for future targets in the development of next-generation biomarkers and therapeutics.

Phase II trial of erlotinib in women with squamous cell carcinoma of the vulva

OBJECTIVE To evaluate the efficacy and toxicity of erlotinib in the management of squamous cell carcinoma (SCC) of the vulva. METHODS Patients with vulvar lesions amenable to surgery or chemoradiation (cohort 1) or those with metastatic measurable disease (cohort 2) received erlotinib 150 mg daily. Patients were monitored for toxicity. Responses were determined by digital photography or RECIST 1.1. Cohort 1 underwent pre and post treatment biopsies. EGFR immunohistochemistry (IHC), fluorescence in-situ hybridization (FISH), and mutational analysis were performed. RESULTS 41 patients were enrolled: 17 in cohort 1 and 24 in cohort 2. Notable grade 3 or 4 toxicities included allergic reaction (1), diarrhea/electrolyte abnormalities (3), ischemic colitis (1), and renal failure (3) and electrolyte abnormalities (n=2). Mean number of cycles for cohort 2 was 3.3. Overall clinical benefit rate was 67.5% with 11 (27.5%) partial responses (PR), 16 (40.0%) stable disease (SD), and 7 (17.5%) progressive disease. Responses were of short duration. All pre and post treatment biopsies exhibited 2-3+ EGFR staining. 5 of 14 patients (35%) were found to have EGFR amplification (n=3) or high polysomy/trisomy (n=2). These five patients had either a PR (n=3) or SD (n=2). Gain of function mutations were not been identified. CONCLUSIONS This is the first reported controlled trial evaluating erlotinib for the management of vulvar carcinoma. Toxicities were acceptable given the lack of treatment options for these patients. Given the observed clinical benefits erlotinib may represent one of the most active agents available to treat vulvar SCC.

5-Hydroxymethylcytosine expression in metastatic melanoma versus nodal nevus in sentinel lymph node biopsies

Sentinel lymph node biopsies are conducted to stage patients with newly diagnosed melanomas that have histopathological attributes conferring defined levels of metastatic potential. Because benign nevic cells may also form ‘deposits’ in lymph nodes (nodal nevus), the pathological evaluation for metastatic melanoma within sentinel lymph nodes can be challenging. Twenty-eight sentinel lymph node biopsy cases containing either metastatic melanoma (N=18) or nodal nevi (N=10) were retrieved from the archives of the Brigham and Women’s Hospital, Department of Pathology (2011–2014). In addition, two sentinel lymph node cases that were favored to represent metastatic disease but whose histopathological features were viewed as equivocal, with melanoma favored, were also included. Dual labeling for the melanocyte lineage marker, MART-1, and the epigenetic marker, 5-hydroxymethylcytosine, a functionally significant indicator that has been shown to distinguish benign nevi from melanoma, was performed on all cases using immunohistochemistry and/or direct immunofluorescence. All (18 of 18) metastatic melanoma cases showed complete loss of 5-hydroxymethylcytosine nuclear staining in MART-1-positive cells, and all (10 of 10) nodal nevus cases demonstrated 5-hydroxymethylcytosine nuclear staining in MART-1-positive cells. In addition, 5-hydroxymethylcytosine staining confirmed the favored diagnoses of metastatic melanoma in the two ‘equivocal’ cases. Thus, 5-hydroxymethylcytosine may be a useful adjunctive marker to distinguish between benign nodal nevi and metastatic melanoma during the evaluation of sentinel lymph node biopsies for metastatic melanoma.

Thoracoabdominal aneurysm repair: anesthetic management.

Surgical repair of descending thoracic and thoracoabdominal aortic aneurysms (TAAs) presents one of the greatest challenges for the anesthesiologist. The challenge comes from the fine balance of complex medical issues in the setting of altered physiology that occurs during the perioperative period. Patients presenting for TAA repair usually have multiple preexisting comorbid conditions involving their cardiac, pulmonary, and renal systems; and aneurysm repair poses a direct and immediate threat to these systems in addition to the gastrointestinal and neurologic systems. To preserve proper function of these organ systems, the anesthesiologist must be adept at monitoring and manipulating rapid and extreme hemodynamic changes, maintaining adequate pulmonary function during one lung ventilation, and preserving metabolic and hematologic homeostasis after aortic crossclamping and unclamping in the setting of significant blood loss. In these high-risk patients, distal aortic perfusion, spinal cord preservation through cooling techniques, distal shunting procedures, and the use of motor-evoked potentials have all been used in attempt to decrease the risk of spinal cord and mesenteric ischemia. The recent development of endovascular stent graft repair is changing the practices of many institutions. Thoracoabdominal aneurysms are classified according to the Crawford classification depending on the extent of the aneurysm. Type I aneurysms extend from the left subclavian artery to the diaphragm. Type II aneurysms extend from the left subclavian artery to below the renal arteries. Type III aneurysms extend from the midthoracic descending aorta to below the

p40 exhibits better specificity than p63 in distinguishing primary skin adnexal carcinomas from cutaneous metastases

The histopathologic distinction between primary adnexal carcinomas and metastatic adenocarcinoma to the skin from sites such as the breast, lung, and others often presents a diagnostic dilemma. Current markers of diagnostic utility include p63 and cytokeratin 5/6; however, their expression has been demonstrated in 11% to 22% and 27% of cutaneous metastases, respectively. Furthermore, the immunoreactivity of p40 and GATA3 in various cutaneous adnexal carcinomas has not been previously reported. In the present study, we compared the expression of p40, p63, cytokeratin 5/6, and GATA3 in a total of 143 cases, including 67 primary adnexal carcinomas and 76 cutaneous metastases. p40, p63, cytokeratin 5/6, and GATA3 expression was observed in 80%, 84%, 86%, and 47% of primary adnexal carcinoma, respectively, and in 8%, 17%, 26%, and 40% of cutaneous metastases, respectively. χ(2) Analysis revealed statistically significant P values (<.0001) for p40, p63, and cytokeratin 5/6 in distinguishing primary adnexal carcinoma from cutaneous metastases. In summary, while p63 and cytokeratin 5/6 have similar sensitivity (84% and 86%, respectively) in detecting primary adnexal carcinomas, p40 appeared to be the most specific marker (92%) with the best positive predictive value (90%). Since breast and lung are the most common sites of origin for cutaneous metastases, p40 is the best distinguishing marker in these settings. None of the four studied markers (p40, p63, cytokeratin 5/6, and GATA3) are helpful in distinguishing between primary adnexal carcinomas from cutaneous metastases of salivary gland or bladder malignancies.

Loss of the epigenetic mark, 5-Hydroxymethylcytosine, correlates with small cell/nevoid subpopulations and assists in microstaging of human melanoma

Melanomas in the vertical growth phase (VGP) not infrequently demonstrate cellular heterogeneity. One commonly encountered subpopulation displays small cell/nevoid morphology. Although its significance remains unknown, such subpopulations may pose diagnostic issues when faced with differentiating such changes from associated nevus or mistaking such regions for nevic maturation (pseudomaturation). That ‘loss’ of the epigenetic biomarker, 5-hydroxymethylcytosine (5-hmC), is a hallmark for melanoma and correlates with virulence prompted us to explore the diagnostic utility and biological implications of 5-hmC immunohistochemistry (IHC) in melanomas with small cell/nevoid subpopulations. Fifty-two cases were included in this study, including melanomas with small cell/nevoid subpopulations (MSCN) or melanomas with pre-existing nevus (MPEN). Semiquantitative and computer-validated immunohistochemical analyses revealed invariable, uniform loss of 5-hmC in the conventional melanoma component. By contrast, the nevic components in MPEN cases demonstrated strong nuclear immunopositivity. In MSCN cases, there was partial to complete loss of 5-hmC restricted to these nevoid areas. Based on recent data supporting tight correlation between 5-hmC loss and malignancy, our findings indicate a potential ‘intermediate’ biological nature for small cell/nevoid subpopulations. Because 5-hmC assisted in differentiating such regions from associated nevus, the use of 5-hmC as an adjunct to microstaging in difficult cases showing VGP heterogeneity should be further explored.

The Structure and Maturation Pathway of Microcin B17

The DNA replication inhibitor microcin B 17 (MccB 17) is a forty-three amino acid peptide antibiotic produced by diverse strains of Escherichia coli (Baquero & Moreno, 1984; Davagnino et al., 1986; Herrero & Moreno, 1986; Vizan et al., 1991). Seven genes, designated mcbABCDEFG, responsible for MccB 17 production and immunity have been found in several large, single-copy plasmids, the most well studied of these being pMccB 17 (San-Millan et al, 1985;Garrido et al., 1988; Genilloud et al., 1989). Four of these genes, mcbABCD, are essential for the production of MccB 17. Initially, we began studying MccB 17 as a small protein amenable to structure/function analysis by using extensive mutagenesis followed by characterization of the mutant proteins. But as we began to characterize MccB 17, we became more and more interested in its unique structure and in the pathway of its synthesis. In this paper we present what we know about the structure of MccB 17. We also present a working model of the maturation pathway of MccB17 and our evidence which supports this model. Lastly, we briefly discuss some interesting observations we have made concerning the regulation of MccB 17 maturation.

Effective use of mirtazapine for refractory pruritus associated with carcinoma en cuirasse

Pruritus is a debilitating symptom that can be associated with cutaneous involvement by an underlying malignancy. We report the case of a 68-year-old woman with a history of triple-negative breast cancer who presented with extensive, localised cutaneous metastasis complicated by incapacitating, treatment-refractory pruritus localised to the anatomic regions involved by her metastatic disease. Physical examination revealed an indurated, ecchymotic, ulcerated plaque circumferentially encasing her thorax. Histopathological examination revealed substantial dermal lymphatic involvement and dilation as well as dermal collagen infiltration by tumour cells and nodules. The clinical and pathological findings were consistent with a diagnosis of carcinoma en cuirasse. Mirtazapine, a noradrenergic antidepressant with antiserotonin and antihistamine activity, was started and led to rapid, sustained relief of the patients pruritus. Mirtazapine may be a useful systemic agent for the palliative relief of pruritus associated with cutaneous infiltration by an underlying malignancy.

Soft tissue angiofibroma: a case report.

Soft tissue angiofibroma is a recently described neoplasm that typically presents as a slowly growing, painless mass in the soft tissues of the lower extremities. Cytogenetic and molecular studies have identified a recurrent t(5;8) translocation. Treatment is simple excision. Existing data suggest that this tumor is benign and has a low rate of local recurrence. The radiologic and pathologic differential diagnoses for this lesion include both benign and malignant lesions, including plantar fibromatosis, tenosynovial giant cell tumor, fibroma of tendon sheath, epithelioid sarcoma, and low-grade myxofibrosarcoma. Proper identification of this benign lesion through radiologic and pathologic correlation is important to prevent misdiagnosis of a low-grade sarcoma.

5-Hydroxymethylcytosine is a Nuclear Biomarker to Assess Biological Potential in Histologically Ambiguous Heavily Pigmented Melanocytic Neoplasms

5‐Hydroxymethylcytosine (5‐hmC) is an epigenetic marker detectable through immunohistochemistry (IHC) that has been shown to distinguish benign nevi from melanoma with high sensitivity and specificity. The purpose of the study was to explore its diagnostic utility in a subset of histologically challenging, heavily pigmented cutaneous melanocytic neoplasms.